RESUMO
The serotonin transporter (SERT) terminates serotonergic signalling through the sodium- and chloride-dependent reuptake of neurotransmitter into presynaptic neurons. SERT is a target for antidepressant and psychostimulant drugs, which block reuptake and prolong neurotransmitter signalling. Here we report X-ray crystallographic structures of human SERT at 3.15 Å resolution bound to the antidepressants (S)-citalopram or paroxetine. Antidepressants lock SERT in an outward-open conformation by lodging in the central binding site, located between transmembrane helices 1, 3, 6, 8 and 10, directly blocking serotonin binding. We further identify the location of an allosteric site in the complex as residing at the periphery of the extracellular vestibule, interposed between extracellular loops 4 and 6 and transmembrane helices 1, 6, 10 and 11. Occupancy of the allosteric site sterically hinders ligand unbinding from the central site, providing an explanation for the action of (S)-citalopram as an allosteric ligand. These structures define the mechanism of antidepressant action in SERT, and provide blueprints for future drug design.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Antidepressivos/química , Antidepressivos/metabolismo , Antidepressivos/farmacologia , Citalopram/química , Citalopram/metabolismo , Citalopram/farmacologia , Cristalografia por Raios X , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Desenho de Fármacos , Espaço Extracelular/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Espaço Intracelular/metabolismo , Íons/química , Íons/metabolismo , Ligantes , Modelos Moleculares , Paroxetina/química , Paroxetina/metabolismo , Paroxetina/farmacologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/imunologia , Relação Estrutura-AtividadeRESUMO
Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel's requirement of cytoplasmic Ca2+ ions for activation.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Canais de Cálcio/química , Ativação do Canal Iônico , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Canais de Cálcio/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Novel methods of postoperative analgesia for total knee arthroplasty (TKA) have demonstrated improved functional outcomes and decreased narcotic consumption. These approaches include continuous adductor canal blocks (CACB) and periarticular injection (PAI). There is a lack of current understanding regarding the effect of these modalities on narcotic usage, functionality, and pain when both PAI and CACB are utilized compared to PAI alone. METHODS: TKAs were performed unilaterally by a single surgeon with a standardized protocol. Patients were divided into two groups: those receiving PAI alone (n = 54) and those receiving PAI and CACB (n = 37). Patient outcomes including, narcotics usage, pain scale, and distance walked, were recorded on postoperative day (POD) zero through three. RESULTS: When compared with PAI alone, it was identified that concurrent use of PAI and CACB results in a statistically significant decrease in narcotics usage on POD 0, 1, 3, and total narcotic usage while admitted. Patients in the PAI and CACB group walked significantly farther than patients in the PAI only group on POD 1, 2, and 3. On POD 0, patients in the PAI and CACB reported significantly less pain with activity when compared to the PAI only group. CONCLUSION: Here we identify an additive effect when utilizing both PAI and CACB for postoperative TKA analgesia. Our findings demonstrate significant decrease in patient total narcotic usage, pain scores, and an increase in walking distance when utilizing PAI and CACB compared with PAI alone. This analgesic technique may help reduce patients' narcotic use while also increasing functional outcomes.
Assuntos
Analgesia/métodos , Anestésicos Locais/administração & dosagem , Artroplastia do Joelho/efeitos adversos , Bupivacaína/administração & dosagem , Bloqueio Nervoso/métodos , Dor Pós-Operatória/prevenção & controle , Idoso , Analgésicos Opioides/uso terapêutico , Terapia Combinada , Feminino , Humanos , Injeções , Lipossomos , Masculino , Pessoa de Meia-Idade , Medição da Dor , Dor Pós-Operatória/etiologia , Período Pós-Operatório , Estudos Retrospectivos , CaminhadaRESUMO
Hemoglobin (Hb) is the heme-containing O2 transport protein essential for life in all vertebrates. The resting high-spin (S = 2) ferrous form, deoxy-Hb, combines with triplet O2, forming diamagnetic (S = 0) oxy-Hb. Understanding this electronic structure is the key first step in understanding transition metal-O2 interaction. However, despite intense spectroscopic and theoretical studies, the electronic structure description of oxy-Hb remains elusive, with at least three different descriptions proposed by Pauling, Weiss, and McClure-Goddard, based on theory, spectroscopy, and crystallography. Here, a combination of X-ray absorption spectroscopy and extended X-ray absorption fine structure, supported by density functional theory calculations, help resolve this debate. X-ray absorption spectroscopy data on solution and crystalline oxy-Hb indicate both geometric and electronic structure differences suggesting that two of the previous descriptions are correct for the Fe-O2 center in oxy-Hb. These results support the multiconfigurational nature of the ground state developed by theoretical results. Additionally, it is shown here that small differences in hydrogen bonding and solvation effects can tune the ground state, tipping it into one of the two probable configurations. These data underscore the importance of solution spectroscopy and show that the electronic structure in the crystalline form may not always reflect the true ground-state description in solution.
Assuntos
Oxiemoglobinas/química , Conformação Proteica , Cristalografia por Raios X , Estrutura Molecular , Espectroscopia por Absorção de Raios X/métodosRESUMO
Multidrug resistance protein 4 (MRP4) is a broadly expressed ATP-binding cassette transporter that is unique among the MRP subfamily for transporting prostanoids, a group of signaling molecules derived from unsaturated fatty acids. To better understand the basis of the substrate selectivity of MRP4, we used cryogenic-electron microscopy to determine six structures of nanodisc-reconstituted MRP4 at various stages throughout its transport cycle. Substrate-bound structures of MRP4 in complex with PGE1, PGE2 and the sulfonated-sterol DHEA-S reveal a common binding site that accommodates a diverse set of organic anions and suggest an allosteric mechanism for substrate-induced enhancement of MRP4 ATPase activity. Our structure of a catalytically compromised MRP4 mutant bound to ATP-Mg2+ is outward-occluded, a conformation previously unobserved in the MRP subfamily and consistent with an alternating-access transport mechanism. Our study provides insights into the endogenous function of this versatile efflux transporter and establishes a basis for MRP4-targeted drug design.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos , Prostaglandinas , Prostaglandinas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transporte Biológico , Dinoprostona/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Early identification and treatment of Staphylococcus aureus (S. aureus) nasal colonization can reduce the risk of prosthetic joint infection. The purpose of this study was to evaluate patient-specific predictors for S. aureus nasal colonization in total joint arthroplasty patients to aid in preoperative screening protocols. A total of 2,147 arthroplasty patients who were preoperatively screened for S. aureus nasal colonization were retrospectively reviewed. Factors analyzed consisted of procedure type, primary diagnosis, gender, ethnicity, body mass index, the presence of chronic obstructive pulmonary disease, obstructive sleep apnea, hypertension, diabetes mellitus, use of immunosuppression medication, smoking history, and chronic kidney disease. Univariate and multivariate analyses were performed with significance p < 0.05 and 95% confidence intervals. Overall, 3.7% (79) of our cohort tested positive for methicillin-resistant Staphylococcus aureus (MRSA), and 23.2% (493) tested positive for methicillin-sensitive Staphylococcus aureus (MSSA). Independent predictors for MRSA colonization were of Hispanic ethnicity (p = 0.001, odds ratio [OR] 13.98, confidence interval [CI] 2.97-65.76), immunosuppression medication use (p = 0.006, OR 2.82, CI 1.35-5.87), and revision total hip arthroplasty (THA) procedure (p < 0.001, OR 7.51, CI 2.58-21.89). Independent predictors for MSSA colonization were body mass index (BMI) >35 (p = 0.002, OR 1.57, CI 1.19-2.1). Variables were found to be protective against MSSA colonization including female gender (p = 0.012, OR 0.76, CI 0.61-0.94), age 60 to 69 (p = 0.025, OR 0.75, CI 0.58-0.96), and age 70 to 79 (p = 0.002, OR 0.63, CI 0.47-0.84). Age, Hispanic ethnicity, gender, revision THA, use of immunosuppression medication, and elevated BMI were independent risk factors for S. aureus nasal colonization.
Assuntos
Artroplastia de Quadril , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Idoso , Artroplastia de Quadril/efeitos adversos , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus , Infecção da Ferida Cirúrgica/etiologiaRESUMO
The trimeric serine protease HTRA1 is a genetic risk factor associated with geographic atrophy (GA), a currently untreatable form of age-related macular degeneration. Here, we describe the allosteric inhibition mechanism of HTRA1 by a clinical Fab fragment, currently being evaluated for GA treatment. Using cryo-EM, X-ray crystallography and biochemical assays we identify the exposed LoopA of HTRA1 as the sole Fab epitope, which is approximately 30 Å away from the active site. The cryo-EM structure of the HTRA1:Fab complex in combination with molecular dynamics simulations revealed that Fab binding to LoopA locks HTRA1 in a non-competent conformational state, incapable of supporting catalysis. Moreover, grafting the HTRA1-LoopA epitope onto HTRA2 and HTRA3 transferred the allosteric inhibition mechanism. This suggests a conserved conformational lock mechanism across the HTRA family and a critical role of LoopA for catalysis, which was supported by the reduced activity of HTRA1-3 upon LoopA deletion or perturbation. This study reveals the long-range inhibition mechanism of the clinical Fab and identifies an essential function of the exposed LoopA for activity of HTRA family proteases.
Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A , Degeneração Macular , Serina Endopeptidases , Cristalografia por Raios X , Epitopos , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.
RESUMO
Viral infection and pathogenesis is mediated by host protein-viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-ß-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12-419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.
Assuntos
HIV-1/metabolismo , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticorpos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Complexos Multiproteicos/metabolismo , Ligação Proteica , Replicação Viral/fisiologiaRESUMO
Antibodies (Abs) are ubiquitous reagents for biological and biochemical research and are rapidly expanding into new therapeutic areas. They are one of the most important probes for determining how proteins function under normal and pathophysiological conditions. Abs are required for the quantification of targets, detection of temporal and spatial patterns of protein expression in cells and tissues, and identification of interacting partners and their biological activities. Their remarkable specificity and unique binding properties can facilitate three-dimensional structure determination using X-ray crystallography and electron cryomicroscopy. While hybridoma technology that involves animal immunization is often productive, many antigen targets do not generate useful Abs. This is particularly true if unique states of the target or critical non-immunogenic target sequences need to be recognized by the Abs. By using the methods of recombinant antibody generation, identification, and engineering, these 'hybridoma-refractory' antigens can be readily targeted. Specific, reproducible, and renewable recombinant Abs are proving to be invaluable reagents in applications ranging from biological discovery to structure determination of challenging macromolecules.
Assuntos
Anticorpos/imunologia , Animais , Imunização , Proteínas RecombinantesRESUMO
A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins-MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.
RESUMO
Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.
RESUMO
The serotonin transporter is a sodium and chloride-coupled transporter that "pumps" extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 Å resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.
Assuntos
Citalopram/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Cristalização , Humanos , Ligação Proteica , Serotonina , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/isolamento & purificação , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de SerotoninaRESUMO
Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.
Assuntos
Análise Mutacional de DNA , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico , Ubiquitina/genética , Ubiquitina/metabolismo , Biologia/educação , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/fisiologia , Estudantes , UniversidadesRESUMO
Serotonin is a ubiquitous chemical transmitter with particularly important roles in the gastrointestinal, cardiovascular and central nervous systems. Modulation of serotonergic signaling occurs, in part, by uptake of the transmitter by the serotonin transporter (SERT). In the brain, SERT is the target for numerous antidepressants including tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs). Despite the importance of SERT in human physiology, biochemical, biophysical and high-resolution structural studies have been hampered due to the instability of SERT in detergent micelles. To identify a human SERT (hSERT) construct suitable for detailed biochemical and structural studies, we developed an efficient thermostability screening protocol and rapidly screened 219 mutations for thermostabilization of hSERT in complex with the SSRI paroxetine. We discovered three mutations-Y110A, I291A and T439S -that, when combined into a single construct, deemed TS3, yielded a hSERT variant with an apparent melting temperature (Tm) 19°C greater than that of the wild-type transporter, albeit with a loss of transport activity. Further investigation yielded a double mutant-I291A and T439S-defined as TS2, with a 12°C increase in Tm and retention of robust transport activity. Both TS2 and TS3 were more stable in short-chain detergents in comparison to the wild-type transporter. This thermostability screening protocol, as well as the specific hSERT variants, will prove useful in studies of other integral membrane receptors and transporters and in the investigation of structure and function relationships in hSERT.
Assuntos
Antidepressivos Tricíclicos/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Substituição de Aminoácidos , Transporte Biológico Ativo , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Estabilidade Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
BACKGROUND: The Canadian Health Measures Survey (CHMS) is an ongoing cross-sectional national survey that includes a measure of 25-hydroxyvitamin D [25(OH)D] by immunoassay. For cycles 1 and 2, the collection period occurred approximately every 2 y, with a new sample of â¼5600 individuals. OBJECTIVE: The goal was to standardize the original 25(OH)D CHMS values in cycles 1 and 2 to the internationally recognized reference measurement procedures (RMPs) developed by the US National Institute for Standards and Technology (NIST) and Ghent University, Belgium. DESIGN: Standardization was accomplished by using a 2-step procedure. First, serum samples corresponding to the original plasma samples were remeasured by using the currently available immunoassay method. Second, 50 serum samples with known 25(OH)D values assigned by the NIST and Ghent reference method laboratories were measured by using the currently available immunoassay method. The mathematical models for each step-i.e., 1) YCurrent = XOriginal and 2) YNIST-Ghent = XCurrent -were estimated by using Deming regression, and the 2 models were solved to obtain a single equation for converting the "original" values to NIST-Ghent RMP values. RESULTS: After standardization (cycles 1 and 2 combined), the percentage of Canadians with 25(OH)D values <40 nmol/L increased from 16.4% (original) to 19.4% (standardized), and values <50 nmol/L increased from 29.0% (original) to 36.8% (standardized). The 25(OH)D standardized distributions (cycles 1 and 2 analyzed separately) were similar across age and sex groups; slightly higher values were associated with cycle 2 in the young and old. This finding contrasts with the original data, which indicated that cycle 2 values were lower for all age groups. CONCLUSION: The shifts in 25(OH)D distribution brought about by standardization indicate its importance in drawing correct conclusions about potential population deficiencies and insufficiencies and in permitting the comparison of distributions between national surveys.
Assuntos
25-Hidroxivitamina D 2/sangue , Calcifediol/sangue , Modelos Estatísticos , Avaliação Nutricional , Deficiência de Vitamina D/diagnóstico , Adolescente , Adulto , Idoso , Automação Laboratorial , Canadá , Criança , Estudos Transversais , Diagnóstico Precoce , Feminino , Humanos , Imunoensaio , Masculino , Pessoa de Meia-Idade , Inquéritos Nutricionais , Valores de Referência , Deficiência de Vitamina D/sangue , Adulto JovemRESUMO
BACKGROUND: Australian national biomonitoring for persistent organic pollutants (POPs) relies upon age-specific pooled serum samples to characterize central tendencies of concentrations but does not provide estimates of upper bound concentrations. This analysis compares population variation from biomonitoring datasets from the US, Canada, Germany, Spain, and Belgium to identify and test patterns potentially useful for estimating population upper bound reference values for the Australian population. METHODS: Arithmetic means and the ratio of the 95th percentile to the arithmetic mean (P95:mean) were assessed by survey for defined age subgroups for three polychlorinated biphenyls (PCBs 138, 153, and 180), hexachlorobenzene (HCB), p,p-dichlorodiphenyldichloroethylene (DDE), 2,2',4,4' tetrabrominated diphenylether (PBDE 47), perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). RESULTS: Arithmetic mean concentrations of each analyte varied widely across surveys and age groups. However, P95:mean ratios differed to a limited extent, with no systematic variation across ages. The average P95:mean ratios were 2.2 for the three PCBs and HCB; 3.0 for DDE; 2.0 and 2.3 for PFOA and PFOS, respectively. The P95:mean ratio for PBDE 47 was more variable among age groups, ranging from 2.7 to 4.8. The average P95:mean ratios accurately estimated age group-specific P95s in the Flemish Environmental Health Survey II and were used to estimate the P95s for the Australian population by age group from the pooled biomonitoring data. CONCLUSIONS: Similar population variation patterns for POPs were observed across multiple surveys, even when absolute concentrations differed widely. These patterns can be used to estimate population upper bounds when only pooled sampling data are available.
Assuntos
Bases de Dados Factuais/normas , Monitoramento Ambiental , Poluentes Ambientais/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácidos Alcanossulfônicos/sangue , Austrália , Caprilatos/sangue , Criança , Pré-Escolar , Poluentes Ambientais/química , Fluorocarbonos/sangue , Humanos , Hidrocarbonetos Clorados/sangue , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Praguicidas/sangue , Bifenilos Policlorados/sangue , Adulto JovemRESUMO
Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.